Method for producing liquid bacterial fertilizer by utilizing ackermania muciniphila fermentation waste liquid

Abstract

The invention discloses a method for producing a liquid bacterial fertilizer by using ackermania muciniphila fermentation waste liquid, which comprises the following steps: adjusting the pH value of the ackermania muciniphila fermentation waste liquid to 6.5-7.5; and putting the mixture into a fermentation tank, sterilizing, cooling to 35-39 DEG C, inoculating a seed solution of at least one of bacillus and lactobacillus according to the volume content of 1-5%, and fermenting for 20-30 hours to obtain the liquid bacterial fertilizer. The method is simple in preparation, the ackermania muciniphila fermentation waste liquid can be turned into wealth, environmental protection burden is relieved, the liquid bacterial fertilizer obtained through the method is low in COD, water is purified, gamma-polyglutamic acid is contained, and when the liquid bacterial fertilizer is applied to plants, the soil structure can be improved, plant root development and plant growth can be promoted, and green low-carbon cycle development is achieved.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation and relates to a method for producing liquid bacterial fertilizer by utilizing fermentation waste liquid of Akkermansia muciniphila. Background Art

[0002] Akkermansia muciniphila, referred to as AKK bacteria, exists naturally in the intestinal wall of healthy people. Studies have shown that AKK bacteria have multiple functions, including enhancing immunity, anti-inflammation, anti-aging, fat reduction, anti-aging, etc. It is also helpful in preventing or improving diabetes, liver disease and cardiovascular disease, and is expected to become the next generation of star probiotics. The commonly used culture medium is mainly BHI culture medium combined with mucin, and a variety of amino acids, growth factors and other carbon and nitrogen sources are added, but the AKK bacteria yield is very low, and the fermentation waste liquid contains a large amount of nutrients and cannot be reused. At present, it can only be treated as industrial wastewater. If the fermentation waste liquid of Akkermansia muciniphila can be utilized and turned into treasure, it will be of great significance to achieve energy conservation and emission reduction and promote green and low-carbon transformation. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for producing liquid bacterial fertilizer by utilizing fermentation waste liquid of Akkermansia muciniphila.

[0004] The technical solution of the present invention is summarized as follows:

[0005] A method for producing liquid bacterial fertilizer by using fermentation waste liquid of Akkermansia muciniphila comprises the following steps: adjusting the pH value of the fermentation waste liquid of Akkermansia muciniphila to 6.5-7.5 with a sodium hydroxide aqueous solution; putting the waste liquid into a fermentation tank, sterilizing it, cooling it to 35-39 DEG C, adding seed liquid of at least one of bacillus and lactobacillus at a volume content of 1%-5%, fermenting it for 20-30 hours, and obtaining liquid bacterial fertilizer.

[0006] The bacillus is at least one of Bacillus Velezii and Bacillus licheniformis.

[0007] The lactobacillus is at least one of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei.

[0008] Advantages of the present invention:

[0009] The method of the present invention is simple to prepare and can not only transform the fermentation waste liquid of Akkermansia muciniphila into treasure and reduce the environmental burden, but also the liquid bacterial fertilizer obtained by the method of the present invention has low COD, purifies water quality, contains gamma-polyglutamic acid, and is applied to plants to improve soil structure, promote plant root development and plant growth, and realize green and low-carbon circular development. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Comparison of COD in liquid bacterial fertilizer and fermentation waste liquid in Examples 6-10.

[0011] Figure 2 is the standard curve of γ-polyglutamic acid.

[0012] Figure 3 Comparison of the content of γ-polyglutamic acid in the liquid bacterial fertilizer and the fermentation waste liquid in Examples 6-10.

[0013] Figure 4 Comparison of tobacco biomass between the experimental group and the control group.

[0014] Figure 5 Comparison of root and stem lengths between the experimental group and the control group.

[0015] Figure 6 Comparison of aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight between the experimental group and the control group. DETAILED DESCRIPTION

[0016] The Akkermansia muciniphila fermentation waste liquid of the present invention is the waste liquid obtained by centrifugation after the fermentation of Akkermansia muciniphila in the laboratory by the inventor of the present invention (containing incompletely utilized BHI culture medium, mucin, amino acids, and other carbon and nitrogen sources and other nutrients), but it is not intended to limit the present invention. The Akkermansia muciniphila is Akkermansia muciniphila AKK-PL001, which is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, deposited on March 14, 2024, and deposited with CCTCC NO: M2024483.

[0017] The present invention will be further described below by means of specific examples. It should be understood that the following examples are only for a better understanding of the present invention, rather than for limiting the scope of the present invention.

[0018] The strains used in the embodiments of the present invention are as follows:

[0019] Bacillus velezensis XW0513 is deposited in the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: July 31, 2020, deposit number: CGMCC No.20474.

[0020] Bacillus licheniformis J6 is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The deposit date is December 4, 2023. The deposit number is CCTCC NO: M20232451.

[0021] Lactobacillus plantarum LPA6, deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, deposit date: October 11, 2023, deposit number: CCTCC NO: M20231856.

[0022] The Lactobacillus rhamnosus was a commercial strain Lactobacillus rhamnosus LR519.

[0023] The Lactobacillus paracasei was a commercial strain of Lactobacillus paracasei LP33.

[0024] Other Bacillus velez, Bacillus licheniformis, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei may also be used in the present invention.

[0025] Example 1

[0026] Preparation of Bacillus Velez seed liquid: Bacillus Velez glycerol bacteria is accessed into LB liquid culture medium at a volume content of 0.5% (0.1%-1% is all right, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), and cultured in a shaking incubator at 37° C. (35-39° C. any value, such as 35° C., 36° C., 38° C., 39° C.), 200 rpm for 14 hours to obtain the Bacillus Velez seed liquid.

[0027] Example 2

[0028] Preparation of Bacillus licheniformis seed solution: Inoculate 0.5% (0.1%-1% by volume, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, 1%) of Bacillus licheniformis glycerol bacteria into LB liquid culture medium, and culture in a shaking incubator at 37° C. (any value of 35-39° C., such as 35° C., 36° C., 38° C., 39° C.), 200 rpm for 14 hours to obtain Bacillus licheniformis seed solution.

[0029] Example 3

[0030] Plant lactobacillus seed liquid preparation: plant lactobacillus glycerol bacteria is accessed in the MRS liquid nutrient medium by volume content 0.5% (0.1%-1% can be, as 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), in shaking table, 37 ℃ (35-39 ℃ arbitrary value, as 35 ℃, 36 ℃, 38 ℃, 39 ℃), 200rpm, cultivated 14 hours, obtained plant lactobacillus seed liquid.

[0031] Example 4

[0032] Preparation of Lactobacillus rhamnosus seed liquid: Lactobacillus rhamnosus glycerol bacteria is accessed into MRS liquid culture medium by volume content 0.5% (0.1%-1% is all right, as 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), in shaking table 37 ℃ (35-39 ℃ arbitrary value, as 35 ℃, 36 ℃, 38 ℃, 39 ℃), 200rpm, cultivated 14 hours, obtained Lactobacillus rhamnosus seed liquid.

[0033] Example 5

[0034] Lactobacillus paracasei seed liquid preparation: Lactobacillus paracasei glycerine bacteria is accessed in the MRS liquid nutrient medium by volume content 0.5% (0.1%-1% can be, as 0.1%, 0.2%, 0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), in shaking table, 37 ℃ (35-39 ℃ arbitrary value, as 35 ℃, 36 ℃, 38 ℃, 39 ℃), 200rpm, cultivated 14 hours, obtained Lactobacillus paracasei seed liquid.

[0035] Example 6

[0036] A method for producing liquid bacterial fertilizer using fermentation waste liquid of Akkermansia muciniphila, comprising the following steps: adjusting the pH value of the fermentation waste liquid of Akkermansia muciniphila to 7.0 with a sodium hydroxide aqueous solution; putting the waste liquid into a fermentation tank, sterilizing it at 115° C. for 30 min, and cooling it to 37° C.; adding the seed liquid prepared in Example 1 at a volume content of 3% (the content of Bacillus velez in the seed liquid is 3×10 9 CFU / ml), fermented for 25 hours to obtain liquid bacterial fertilizer.

[0037] Example 7

[0038] A method for producing liquid fertilizer by using fermentation waste liquid of Akkermansia muciniphila, comprising the following steps: adjusting the pH value of the fermentation waste liquid of Akkermansia muciniphila to 6.5 with a sodium hydroxide aqueous solution; putting the waste liquid into a fermentation tank, sterilizing it at 121°C for 20 minutes, and cooling it to 35°C; adding seed liquid (Bacillus velez and Lactobacillus plantarum in a volume ratio of 1:1, with the contents of 0.5×10 9 CFU / ml, 5×10 9 CFU / ml), ferment for 20 hours to obtain liquid bacterial fertilizer.

[0039] Example 8

[0040] A method for producing liquid fertilizer using fermentation waste liquid of Akkermansia muciniphila, comprising the following steps: adjusting the pH value of the fermentation waste liquid of Akkermansia muciniphila to 7.5 with a sodium hydroxide aqueous solution; putting the waste liquid into a fermentation tank, sterilizing it at 118°C for 30 minutes, and cooling it to 39°C; adding seed liquid at a volume content of 1% (the content of Lactobacillus plantarum in the seed liquid is 3×10 9 CFU / ml), ferment for 30 hours to obtain liquid bacterial fertilizer.

[0041] Example 9

[0042] A method for producing liquid fertilizer by using fermentation waste liquid of Akkermansia muciniphila, comprising the following steps: adjusting the pH value of the fermentation waste liquid of Akkermansia muciniphila to 6.8 with a sodium hydroxide aqueous solution; putting the waste liquid into a fermentation tank, sterilizing it at 116°C for 30 minutes, and cooling it to 36°C; adding seed liquid (Bacillus velezensis and Bacillus licheniformis in a volume ratio of 1:1, with the contents of 5×10 9 CFU / ml, 5×10 9 CFU / ml), ferment for 22 hours to obtain liquid bacterial fertilizer.

[0043] Example 10

[0044] A method for producing liquid bacterial fertilizer by using fermentation waste liquid of Akkermansia muciniphila, comprising the following steps: adjusting the pH value of the fermentation waste liquid of Akkermansia muciniphila to 7.2 with a sodium hydroxide aqueous solution; putting the waste liquid into a fermentation tank, sterilizing it at 120°C for 30 minutes, and cooling it to 38°C; inoculating a seed solution with a volume content of 4% (Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei in a volume ratio of 1:1:1, with the contents of 5×10 9 CFU / ml, 5×10 9 CFU / ml, 5×10 9 CFU / ml), ferment for 28 hours to obtain liquid bacterial fertilizer.

[0045] Embodiment 11

[0046] COD detection in the fermentation waste liquid of Akkermansia muciniphila (hereinafter referred to as fermentation waste liquid) and the liquid bacterial fertilizer prepared in Examples 6-10.

[0047] The COD content was detected by rapid digestion spectrophotometry. The fermentation waste liquid and liquid bacterial fertilizer were diluted 40 times with water, and 2 mL of each was placed in the digester DRB200 and digested at 150°C. The COD in the liquid was detected by Hach spectrophotometer DR6000. The results were as follows: Figure 1 As shown, the COD content in the fermentation waste liquid is 32640 mg / L, and the COD content in the liquid fertilizer prepared in Examples 6-10 is below 7200 mg / L, indicating that the COD is significantly reduced by the method of the present invention, and the water quality of the fermentation waste liquid is improved.

[0048] Example 12

[0049] Detection of γ-polyglutamic acid content in the fermentation waste liquid and the liquid bacterial fertilizer prepared in Examples 6-10.

[0050] γ-Polyglutamic acid is a special anionic natural polymer with environmental friendliness, super strong chelating adsorption capacity, super high water absorption and moisture retention capacity and good biodegradability. It can balance soil pH, retain water and fertilizer, improve soil structure, and complex harmful heavy metals. The γ-Polyglutamic acid produced in the liquid bacterial fertilizer of the present invention has a good promoting effect on seed germination and plant growth.

[0051] Accurately weigh 0.0100g of γ-polyglutamic acid standard, dilute to 50mL with sterile water, and prepare 200mg / L γ-polyglutamic acid mother solution. Dilute the mother solution with sterile water to prepare 5, 10, 15, 20, 30, 40, 50mg / L γ-polyglutamic acid aqueous solutions, respectively, and mix with 2g / L hexadecyltrimethylammonium bromide (CTAB) solution (prepared with 5% NaOH aqueous solution) in a ratio of 1:1, react for 3min, measure its absorbance at a wavelength of 250nm, and draw a standard curve with γ-polyglutamic acid concentration as the horizontal axis and absorbance as the vertical axis ( Figure 2 ).

[0052] Determination of γ-polyglutamic acid. Take the fermentation waste liquid and the liquid fertilizer prepared in Example 6-10 and dilute them 10 times with sterile water respectively. Take 2 mL of the diluted liquid fertilizer and add it to a centrifuge tube. Centrifuge at a speed of 12000r / min for 3 minutes. Take the supernatant and add an equal volume of CTAB sodium hydroxide solution to react to form a suspension. Complex for 3 minutes, measure the absorbance at a wavelength of 250nm, and bring it into the standard curve to obtain the actual content. The results are as follows Figure 3As shown, the content of γ-polyglutamic acid in the fermentation waste liquid was detected to be 0.14 mg / L, which is extremely low. The content of γ-polyglutamic acid in the liquid fertilizer obtained in Examples 6-10 was all above 370 mg / L, with the highest reaching 440.07 mg / L. This indicates that the liquid fertilizer contains a relatively high concentration of γ-polyglutamic acid using the method of the present invention.

[0053] Example 13

[0054] Liquid bacterial fertilizer promotes plant root development and plant growth.

[0055] 1 Tobacco seed germination experiment

[0056] Take tobacco seeds (Nicotiana benthamiana) of uniform size and sterilize them with 75% ethanol aqueous solution for 1-2 minutes, rinse them with sterile water for 4 times, blow the seeds evenly into the soil of a small pot with a pipette, cover with plastic wrap, and let it stand for germination. When the tobacco seeds grow 2-3 leaves, transplant the tobacco plants, carefully pull out the tobacco with roots with tweezers, and transplant them into a new small pot with sterilized soil, with 1-2 tobacco seedlings transplanted per pot.

[0057] 2Liquid bacterial fertilizer growth promotion experiment

[0058] After transplanting tobacco 7d, the tobacco seedlings of uniform growth were fertilized, and the liquid fertilizer (experimental group) produced in Example 6 was diluted 10 times with sterile water, and 20mL was watered at every pot of seedling root, and the control group was treated with equal amount of sterile water, and irrigated for the second time every 7d, and the method dosage was identical, and irrigated 2 times in total. To ensure saline-alkali conditions, after each irrigation 3d, 20mL 100mM NaHCO3 aqueous solution was watered to every pot of seedlings, and the seedling growth was observed, and 20mL of sterile water was poured every 3d for every pot of seedlings.

[0059] 3. Tobacco seedling biomass determination

[0060] 45 days after the second application of liquid fertilizer, the tobacco seedlings with different treatments were slowly poured out of the pots with roots, and the soil around the roots of the seedlings was slowly washed with running water. After cleaning, the stem length and root length of the tobacco seedlings were measured ( Figure 4 ), and then measured the underground fresh weight (root fresh weight), aboveground fresh weight (stem fresh weight and leaf fresh weight), underground dry weight (root dry weight), and aboveground dry weight (stem dry weight and leaf dry weight). The results showed that under 100mM NaHCO3 stress, the stem length, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of the experimental group were significantly increased compared with the control group ( Figure 5 and Figure 6), its stem length and root length increased by 55.79% and 15.19% respectively, and the aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight increased by 2.3 times, 1.2 times, 1.3 times and 7.9 times respectively, indicating that liquid bacterial fertilizer has a strong growth-promoting ability on plants.

[0061] Experiments have shown that the effects of the liquid bacterial fertilizer produced in Examples 7-10 on promoting plant root development and plant growth are similar to those of the liquid bacterial fertilizer produced in Example 6.

[0062] Results Analysis

[0063] The method of the invention can transform waste liquid from fermentation of Akkermansia muciniphila into treasure, reduce the environmental burden, and produce valuable bacterial fertilizer. The bacterial fertilizer has low COD, purifies water quality, contains gamma-polyglutamic acid, and can improve soil structure and promote plant growth.

Claims

1. A method for producing liquid fertilizer using fermentation waste liquid of Akkermansia muciniphila, characterized in that The method comprises the following steps: adjusting the pH value of fermentation waste liquid of Akkermansia muciniphila to 6.5-7.5; putting the waste liquid into a fermentation tank, sterilizing it, cooling it to 35-39 DEG C, adding seed liquid of at least one of Bacillus and Lactobacillus at a volume content of 1%-5%, fermenting it for 20-30 hours, and obtaining liquid bacterial fertilizer.

2. The method according to claim 1, characterized in that The bacillus is at least one of Bacillus Velezii and Bacillus licheniformis.

3. The method according to claim 1, characterized in that The lactobacillus is at least one of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei.

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